This project is directed toward identifying the actual products of chemical reaction between alkylating agents which are mutagens and/or carcinogens and informational macromolecules, and then assessing the role of various modifications in producing biological effects. Specifically we plan to study the products formed and their fate during replication, when the powerful carcinogen, ethylnitrosourea, acts on DNA in proliferating normal and xeroderma pigmentosum fibroblasts and on fetal rat brain and rat liver cells. We know from our recent work that N-nitroso ethylating agents primarily alkylate oxygens (in vitro and in vivo), including the phosphodiesters and base oxygens of C, U, T and G (and in the case of RNA, the 2'-O of ribose) and several of these are mutagenic events. The effects of oxygen substitution on the physical and biological properties of nucleic acids will be studied in several ways. After determining the stability of O-alkyl derivatives under a variety of conditions, we have developed appropriate methods for quantitating all the O-alkyl bases, 7-EtG, 3-EtG and ethyl-phosphotriesters in enzyme digests of 14C-ethyl-nitrosourea-treated DNA with a total of 2-6 ethyl groups per 10 to the 5th power DNA-P. At this level of alkylation, the cells show little cytotoxicity and can be replicated so that excision of each alkyl derivative can also be quantitatively determined. Our objective is to find whether there is a correlation between rates of excision or persistence of O-alkyl products and the biological effects observed with cells lacking UV repair enzymes (xeroderma pigmentosum fibroblasts) or susceptible to neoplastic transformation (fetal rat brain cells). We will also attempt to prepare homopolymers of O-alkyl pyrimidines in order to study their secondary structure, transcription and translation. The effect of O-alkyl substitution in translation will also be studied by examining the ability of modified codons to function in the binding of tRNA in the presence of ribosomes, or the ability of modified mRNA to be translated accurately in a wheat embryo cell-free system.